Protein O-GlcNAcylation (N-acetylglucosamine modification), which decreases phosphorylation, plays a critical role in cell-cycle regulation, apoptosis and signal transduction. So far, ~120 O-GlcNAcylated human proteins have been found. To elucidate functions of O-GlcNAcylation, it is necessary to develop site-specific antibodies for O-GlcNAcylated proteins. Many pan-specific antibodies for O-GlcNAcylation are available. However, few site-specific antibodies for O-GlcNAcylated proteins have been successfully produced. During the Phase I study, we will synthesize multiple antigen peptides with various features against O-GlcNAcylation sites of c-myc and p53 to test effects of a specific peptide design on the sensitivity and specificity of the antibodies. Sensitivity and specificity of the antibodies will be determined to find out the best peptide design. We will explore utility of the O-GlcNAcylation site-specific antibodies using human breast cell lines, normal (MCF10A) and tumor cells (MCF-7 and N-ras and H-ras MCF10A) with and without treatment with various O-GlcNAcase inhibitors.